N-acetylserotonrn (NAS) is synthesized from serotonin by an action of serotonin N-acetyltransferase (AANAT). Although evidence of hippocampal and cerebellar localization/synthesis of NAS was provided several years ago, it is still believed that in mammals, AANAT is almost exclusively expressed in the pmeal gland and in the retina, and that its neuronal localization is restricted to the lower organisms, such as the fruit fly. We recently reported that AANAT mRNA is expressed in the rat hippocampus and that it is up-regulated by the antidepressant fluoxetine (administered chronically); others found that NAS possesses antidepressant-like activity in a rodent behavioral despair test. Furthermore, we a) identified those neurons in the rat CNS that express AANAT mRNA, including cerebellar granule neurons (CON), b) established primary CON cultures in which AANAT mRNA is normally expressed and is up-regulated by isoproterenol, c) demonstrated that CON cultures treated with 3H-labeled serotonin synthesize NAS, and d) found that in these neurons NAS may act as a functional inhibitor of nitric oxide (NO) synthesis. We hypothesize that NAS can affect neuronal functioning via the inhibition of NO synthase (NOS) activity and/or expression (possibly by an action on the synthesis of the NOS cofactor tetrahydrobiopterin BH4), and that neuronal expression of AANAT could be a target for the action of antidepressant treatments. These hypotheses will be tested in the following 6 AIMs: (1) Characterize in primary rat CON cultures andin BV-2 microglial cultures the pathways involved in NAS-triggered inhibition of NOS, including the synthesis of tetrahydrobiopterin, BH4; (2) Characterize in vitro the role of AANAT in metabolizing serotonin into NAS and in the synthesis of BH4 and/or NOS using cultures from AANAT mutated mice (expressing enzymatically inactive AANAT) and normal mice; (3) Characterize in AANAT-mutated and normal mice the basal and the stimulated BH4 and NO synthesis; (4) Characterize in rat CON cultures the neurotransmitter systems capable of regulating AANAT expression; (5) Investigate in rats whether antidepressants other than fluoxetine increase the brain content of AANAT mRNA, and whether all brain regions expressing AANAT and the pineal glands are equally affected; (6) Characterize whether the AANAT-mutated mice respond to antidepressant differently from normal mice in a model of forced swimming. Techniques to be used include: quantitative reverse transcription/polymerase chain reaction (RT-PCR) and in situ RT-PCR for AANAT mRNA; assays of NOS activity, nitrite and BH4 contents; and the forced swimming test. We expect the results to elucidate the role of AANAT/NAS in neuronal functioning, and in the long term, in the pathobiology of depression.